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(Restrictionendonucleases). These have the ability to cut DNA molecules at very precise sequences of 4 to 8 base

pairscalledrecognition sites. These enzymes are the “molecular scissors” that allow genetic engineers to cut upDNAin a controlled way. First isolated in 1970, these enzymes were discovered earlier in many bacteria.The purified forms of these bacterial restriction enzymes are used today as tools to cut DNA. Restrictionenzymes are named according to the species they were first isolated from, followed by a number todistinguish different enzymes isolated from the same organism.It was observed that certain bacteriophages(viruses)could not infect bacteria other than their usual hosts. It was found the reason for this was thatother potential hosts could destroy almost all of the phage DNA using restriction enzymes present naturallyin their cells; adefense mechanism against the entry of foreign DNA.By using this molecular tool kit, ofover 400 restriction enzymes recognizing about 100 recognition sites, genetic engineers can isolate, sequence,and manipulate individual genes derived from any type of organism. The sites at which the fragments ofDNA are cut may result in overhanging “sticky ends” or non-overhanging “blunt ends”. Pieces may later bejoined together using an enzyme calledDNA ligase

(molecular glue) in a process called

ligation

(gluing).

Recognitions Sites for Selected Restriction Enzymes

Enzyme

Source

Recognition Sites

EcoRI

Escherichia coli

RY-13

G

/

A A T T C

BamHI

Bacillus amyloliquefaciens

H

G

/G A T C C

HaeIII

Haemophilus aegyptius

G G

/

C C

HindIII

Haemophilus influenzae

Rd

A

/

A G C T T

HpaI

Haemophilus parainfluenzae

G T T/A A C

HpaII

Haemophilus parainfluenzae

C C/G G

MboI

Moraxella bovis

/G A T C

NotI

Norcardia otitidis-caviarum

G C/G G C C G C

TaqI

SmaI

Thermus aquaticus

Serratia marcescans

T/C G A

C C C/ G G G

1.

Define what a restriction enzyme is

2.

What is a recognition site.

3.

Differentiate between a sticky end and a blunt end.

4.

The action of a specific sticky end restriction enzyme is illustrated below. Use the table on the prior page to:

a.

Name the restriction enzymes

used:______________________________________________________

b.

Name the organisms

from which

they were

first isolated:

____________________________________

c.

State the base sequence for this restriction enzymes’

recognition sites:

__________________________

d.

Identify whether they form sticky or blunt ends: ____________________________________________

5. A genetic engineer wants to use the restriction enzymeBamHI

to cut the DNA sequence below:

a.Consult the table above and state the recognition site for this enzyme: ______________________

b.Place a mark at each point where the restriction enzymeBamHI would cleave the DNA sequencebelow:

Section of a

single strand of DNA of only 300 base pairs

10 20 30 40

50 60

AATGGGTACG/CACAGTGGAT/CCACGTAGTA/TGCGATGCGT/AGTGTTTATG/GAGAGAAGAA/

70 80

90 100 110 120

AACGCGTCGC/CTTTTATCGA/TGCTGTACGG/ATGCGGAAGT/GGCGATGAGG/ATCCATGCAA/

130 140 150 160

170

180

TCGCGGCCGA/TCGCGTAATA/TATCGTGGCT/GCGTTTATTA/TCGTGACTAG/TAGCAGTATG/

190 200 210 220

230

240

CGATGTGACT/GATGCTATGC/TGACTATGCT/ATGTTTTTAT/GCTGGATCCA/GCGTAAGCAT/

250 260 270 280

290

300

TTCGCTGCGT/GGATCCCATA/TCCTTATATG/CATATATTCT/TATACGGATC/GCGCACGTTT

c.State how many times the DNA was cut by the restriction enzymes: _______________________

d.State how many fragments of DNA were created by this action:

__________________________

e. What were the lengths of each DNA fragment in base pairs: ____________

____________

____________

____________

____________

____________

Gel Electrophoresis

Background Information:

Gel electrophoresis

is a method that separated large molecules (including nucleic acids

or proteins) on thebasis of size, electric charge, and other physical properties. Such molecules possess a slight electric charge.In the case of DNA, DNA has a negative charge. To prepare DNA for gel electrophoresis, the DNA is oftencut up into smaller pieces. This is done by mixing DNA with restriction enzymes in controlled conditions forabout an hour. Calledrestriction digestion, it produces a range of DNA fragments of different lengths.During electrophoresis, molecules are forced to move through the pores of a gel calledagarose, when theelectrical current is applied. Active electrodes at each end of the gel provide the driving force. The electricalcurrent from one electrode repels the molecules (theanion

or negative charge)

while the other electrode(cation or positive charge) pulls the fragments towards it. The frictional force of the agarose resists the flowof the molecules,

separating them by size. The rate of migration through the gel is dependant on thestrength of the electrical field, size and shape of the molecules, and on the ionic strength and temperature ofthe buffer in which the moleculesare moving through. In order to visualize the DNA fragments, staining isnecessary. The stained separated molecules in each lane can be seen as a series of bands spread from oneend of the gel to the other. The large fragments will be trapped quickly inthe agarose and will not migrateas far as the shorter DNA fragments.Gel electrophoresis acts like a “molecular sieve”.The size of thefragments can be compared toDNA markers

which are a mixture of DNA molecules of known molecularweights (sizes) which

are also run through the gel.

1. Explain the purpose of gel electrophoresis:

2. Describe the two forces that control the speed at which fragments pass through the gel:

3. Explain why the smallest fragments travel through the gel the fastest:

4.The box below represents a gel during electrophoresis. You are to pretend that you have loaded and run your

gel. ADNA marker lane is given to you in Lane #1. InLane #2, runundigested (uncut)

DNA through it. In

Lane #3, run your digested (cut) DNA.

#1 #2 #3

(-)

300bp

250bp

200bp

150bp

100bp

50bp

25bp

(+)

Ligation

Background Information:

DNA fragments produced using restriction enzymes may be reassembled by a process known asligation.

Both the desired DNA fragment and the DNA that it will be inserted into must be cut with the samerestriction enzyme.

The pieces arethenjoined together using an enzyme known asDNA ligase.DNA ligaseacts as “molecular glue”.DNA of different origins produced in this way is calledrecombinant DNA

(becauseit is DNA that has been recombined from different sources). The

combined techniques of using restrictionenzymes and ligation are the basic tools of genetic engineering.

If the DNA that is inserted in is from adifferent species of organisms, then the organism is said to betransgenic.

1.Explain the two main steps in the process of joining two DNA fragments together:

a. Annealing:

b.

DNA ligase:

c.

Why it necessary to use the same restriction enzyme for both fragments:

2. Explain why ligation is the reverse of the restriction enzyme process:

1.

If two pieces of DNA are cut by thesame restriction enzyme, they willproduce fragments with matchingsticky ends

2.

When two such matching sticky endscome together, they can join by basepairing. This process is calledannealing.This can allow DNAfragments from a different source,perhaps aplasmid, to be joined tothe DNA fragment.

3.

The joined fragments will usuallyform either a linear molecule or acircular one, as shown here for aplasmid. However, othercombinations of fragments canoccur.

4.

The fragments of DNA are joinedtogether by the enzymeDNA ligase,producing a molecule ofrecombinant DNA.